Zaijun Zhang

Baicalein Protects against 6‑OHDA-Induced Neurotoxicity through Activation of Keap1/Nrf2/HO‑1 and Involving PKCα and PI3K/AKT Signaling Pathways

Baicalein, one of the major flavonoids found in Scutellaria baicalensis Georgi, displays neuroprotective effects on experimental models of Parkinsons disease (PD) in vitro and in vivo. Although the antioxidative and/or anti-inflammatory activity of baicalein likely contributes to these neuroprotective effects, other modes of action remain largely uncharacterized. In the present study, baicalein pretreatment significantly prevented cells from 6-hydroxydopamine (6-OHDA)-induced damage by attenuating cellular apoptosis. However, post-treatment with baicalein did not show any restorative effect against 6-OHDA-induced cellular damage. We found that baicalein increased transcriptional factor NF-E2-related factor 2 (Nrf2)/hemo oxygenase 1(HO-1) protein expression and decreased Kelch-like ECH-associated protein 1 (Keap1) in a time- and concentration-dependent manner in PC12 cells. In addition, baicalein induced Nrf2 nuclear translocation and enhanced antioxidant response element (ARE) transcriptional activity, which conferred cytoprotection against 6-OHDA-induced oxidative injury. Moreover, we demonstrated that cytoprotective effects of baicalein could be attenuated by Nrf2 siRNA transfection and the HO-1 inhibitor zinc protoporphyrin (Znpp) as well as the proteasome inhibitor MG132. Furthermore, PKCα and AKT protein phosphorylation were up-regulated by baicalein pretreatment, and selective inhibitors targeted to PKC, PI3K, and AKT could block the cytoprotective effects of baicalein. Taken together, our results indicate that baicalein prevented PC12 cells from 6-OHDA-induced oxidative damage via the activation of Keap1/Nrf2/HO-1, and it also involves the PKCα and PI3K/AKT signaling pathway. Ultimately, the neuroprotective effects of baicalein may endue baicalein as a promising candidate for the prevention of PD.

Hypoglycemic and beta cell protective effects of andrographolide analogue for diabetes treatment

BackgroundWhile all anti-diabetic agents can decrease blood glucose level directly or indirectly, few are able to protect and preserve both pancreatic beta cell mass and their insulin-secreting functions. Thus, there is an urgent need to find an agent or combination of agents that can lower blood glucose and preserve pancreatic beta cells at the same time. Herein, we report a dual-functional andrographolide-lipoic acid conjugate (AL-1). The anti-diabetic and beta cell protective activities of this novel andrographolide-lipoic acid conjugate were investigated.MethodsIn alloxan-treated mice (a model of type 1 diabetes), drugs were administered orally once daily for 6 days post-alloxan treatment. Fasting blood glucose and serum insulin were determined. Pathologic and immunohistochemical analysis of pancreatic islets were performed. Translocation of glucose transporter subtype 4 in soleus muscle was detected by western blot. In RIN-m cells in vitro, the effect of AL-1 on H2O2-induced damage and reactive oxidative species production stimulated by high glucose and glibenclamide were measured. Inhibition of nuclear factor kappa B (NF-κB) activation induced by IL-1β and IFN-γ was investigated.ResultsIn alloxan-induced diabetic mouse model, AL-1 lowered blood glucose, increased insulin and prevented loss of beta cells and their dysfunction, stimulated glucose transport protein subtype 4 (GLUT4) membrane translocation in soleus muscles. Pretreatment of RIN-m cells with AL-1 prevented H2O2-induced cellular damage, quenched glucose and glibenclamide-stimulated reactive oxidative species production, and inhibited cytokine-stimulated NF-κB activation.ConclusionWe have demonstrated that AL-1 had both hypoglycemic and beta cell protective effects which translated into antioxidant and NF-κB inhibitory activity. AL-1 is a potential new anti-diabetic agent.

Synthesis and evaluation of antibacterial activities of andrographolide analogues

Andrographolide (Andro), the main active component of the herb Andrographis paniculata, has been used for many years to treat a variety of diseases including bacterial and viral infections. Andro was recently reported to act by inhibiting the bacterial quorum sensing system. We have synthesized several Andro analogues and investigated their antibacterial activity and mechanism of action. The new compounds were found to be much more potent than the parent Andro in inhibiting bacterial growth and quorum sensing system. Compounds 5 and 7 significantly reduced virulence factor production. Compound 7 completely inhibited Pseudomonas aeruginosa (P. aeruginosa) biofilm formation, and exhibited synergistic activity with conventional antibiotics. These findings suggest that compound 7 may be the basis for future drug development to combat the unmet needs of virulence factor production, biofilm formation and antibiotic resistance.

Antioxidative and thrombolytic TMP nitrone for treatment of ischemic stroke

Ischemic stroke results from brain blood vessel blockage by thrombus, and produces neuronal cell damage and death. While thrombolytic therapy with tPA has achieved some success in clinic, the strategy of using neuroprotective agents to treat ischemic stroke has been disappointing thus far. In the present work, we synthesized TBN, a derivative of the clinically useful stroke drug TMP armed with a powerful free radical-scavenging nitrone moiety. TBN retains the thrombolytic activity of the parent TMP and possesses strong antioxidative properties. TBN demonstrates significant activity in the rat MCAo stroke model. The results suggest that design of molecules possessing both thrombolytic and neuroprotective properties may be a novel strategy for effective stroke therapeutics.

Ethanolic extract of fructus alpinia oxyphylla protects against 6-hydroxydopamine-induced damage of PC12 cells in vitro and dopaminergic neurons in zebrafish

In an attempt to understand the neuroprotective effect of Fructus Alpinia oxyphylla (AOE) and to elucidate its underlying mechanism of action, the ethanolic extract of AOE was investigated using zebrafish and PC12 cell models. AOE prevented and restored 6-hydroxydopamine (6-OHDA)-induced dopaminergic (DA) neuron degeneration and attenuated a deficit of locomotor activity in a zebrafish (Danio rerio) model of Parkinson’s disease (PD). Treatment with AOE increased the viability of 6-OHDA-treated PC12 cells in vitro in a dose-dependent manner by attenuating cellular apoptosis. However, protocatechuic acid (PCA) and chrysin, two known polyphenol components of AOE, could not reproduce the neuroprotective activity of AOE in the PD zebrafish or PC12 cell models. A mechanistic study found that the protective effect of AOE against 6-OHDA-induced neuronal injury involved anti-inflammatory action (down-regulation of gene expression of IL-1β and TNF-α) and anti-oxidative action (inhibition of NO production and iNOS expression in PC12 cells). Moreover, the PI3K-AKT pathway might be part of the mechanism of neuroprotection of AOE. The results of this research are expected to provide a scientific rationale for the use of AOE in the treatment of PD. However, it is important that the active components that contribute to the neuroprotective action of AOE are identified and characterized.

Resveratrol derivative, trans-3,5,4'-trimethoxystilbene, exerts antiangiogenic and vascular-disrupting effects in zebrafish through the downregulation of VEGFR2 and cell-cycle modulation.

Angiogenesis plays an important role in the development of neoplastic diseases such as cancer. Resveratrol and its derivatives exert antiangiogenic effects, but the mechanisms of their actions remain unclear. The aim of this study was to evaluate the antiangiogenic activity of resveratrol and its derivative trans‐3,5,4′‐trimethoxystilbene in vitro using human umbilical vein endothelial cells (HUVECs) and in vivo using transgenic zebrafish, and to clarify their mechanisms of action in zebrafish by gene expression analysis of the vascular endothelial growth factor (VEGF) receptor (VEGFR2/KDR) and cell‐cycle analysis. trans‐3,5,4′‐Trimethoxystilbene showed significantly more potent antiangiogenic activity than that of resveratrol in both assays. In zebrafish, trans‐3,5,4′‐trimethoxystilbene caused intersegmental vessel regression and downregulated VEGFR2 mRNA expression. Trans‐3,5,4′‐trimethoxystilbene also induced G2/M cell‐cycle arrest, most specifically in endothelial cells of zebrafish embryos. We propose that the antiangiogenic and vascular‐targeting activities of trans‐3,5,4′‐trimethoxystilbene result from the downregulation of VEGFR2 expression and cell‐cycle arrest at G2/M phase. J. Cell. Biochem. 109: 339–346, 2010.

Examining the neuroprotective effects of protocatechuic acid and chrysin on in vitro and in vivo models of Parkinson disease.

Polypharmacology-based strategies using drug combinations with different mechanisms of action are gaining increasing attention as a novel methodology to discover potentially innovative medicines for neurodegenerative disorders. We used this approach to examine the combined neuroprotective effects of two polyphenols, protocatechuic acid (PCA) and chrysin, identified from the fruits of Alpinia oxyphylla. Our results demonstrated synergistic neuroprotective effects, with chrysin enhancing the protective effects of PCA, resulting in greater cell viability and decreased lactate dehydrogenase release from 6-hydroxydopamine-treated PC12 cells. Their combination also significantly attenuated chemically induced dopaminergic neuron loss in both zebrafish and mice. We examined the molecular mechanisms underlying these collective cytoprotective effects through proteomic analysis of treated PC12 cells, resulting in the identification of 12 regulated proteins. Two were further characterized, leading to the determination that pretreatment with PCA and chrysin resulted in (i) increased nuclear factor-erythroid 2-related factor 2 protein expression and transcriptional activity; (ii) modulation of cellular redox status with the upregulated expression of hallmark antioxidant enzymes, including heme oxygenase-1, superoxide dismutase, and catalase; and (iii) decreased levels of malondialdehyde, a known lipid peroxidation product. Treatment with PCA and chrysin also inhibited activation of nuclear factor-κB and expression of inducible nitric oxide synthase. Our findings suggest that natural products, when used in combination, can be effective potential therapeutic agents for treating diseases such as Parkinson disease. A therapy involving both PCA and chrysin exhibits its enhanced neuroprotective effects through a combination of cellular mechanisms: antioxidant cytoprotection and anti-inflammation.

A novel Danshensu derivative confers cardioprotection via PI3K/Akt and Nrf2 pathways

BACKGROUND Danshensu (3-(3,4-dihydroxyphenyl) lactic acid, DSS) is one of the most promising cardioprotective components in the root of Salvia miltiorrhiza but its poor chemical stability poses hurdles in its therapeutic development. It is therefore desirable to enhance the stability of DSS by chemical modification to improve its activities. In the present study, a novel DSS derivative named ADTM was synthesized and characterized for its cardioprotective properties. METHODS Oxidative stress was induced in H9c2 cardiomyoblast cells by tert-butylhydroperoxide (t-BHP) and the protective effects of ADTM were evaluated. For in vivo study, adult rats were treated with vehicle, DSS, ADTM or amlodipine (n=6-8/group) for 24h before the induction of acute myocardial ischemia. At the end of each experiment, infarct size was measured. Underlying the mechanisms of the cardioprotective effects of ADTM were further investigated in H9c2 cells and rat myocardium by evaluating the effects of Nrf2 (NF-E2-related factor 2) and Akt/PI3K pathways. RESULTS ADTM was approximately 10 times more effective than DSS against t-BHP-induced cell injury in H9c2 cells. In rat myocardial ischemia model, ADTM treatment significantly alleviated myocardial infarction. Akt/PI3K and Nrf2 pathways were demonstrated to be involved in both in vitro and in vivo experiments. CONCLUSIONS These results demonstrated that ADTM displayed much better cardioprotective effects than its parent compounds both in vitro and in vivo. This cardioprotection is mediated, at least in part, through Akt/PI3K and Nrf2 pathways. This novel compound represents a promising candidate for the treatment of cardiovascular diseases (CVDs), particularly myocardial infarction.

Unexpected neuronal protection of SU5416 against 1- methyl-4-phenylpyridinium ion-induced toxicity via inhibiting neuronal nitric oxide synthase

SU5416 was originally designed as a potent and selective inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) for cancer therapy. In this study, we have found for the first time that SU5416 unexpectedly prevented 1-methyl-4-phenylpyridinium ion (MPP+)-induced neuronal apoptosis in cerebellar granule neurons, and decreased 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons and impairment of swimming behavior in zebrafish in a concentration-dependent manner. However, VEGFR-2 kinase inhibitor II, another specific VEGFR-2 inhibitor, failed to reverse neurotoxicity at the concentration exhibiting anti-angiogenic activity, strongly suggesting that the neuroprotective effect of SU5416 is independent from its anti-angiogenic action. SU5416 potently reversed MPP+-increased intracellular nitric oxide level with an efficacy similar to 7-nitroindazole, a specific neuronal nitric oxide synthase (nNOS) inhibitor. Western blotting analysis showed that SU5416 reduced the elevation of nNOS protein expression induced by MPP+. Furthermore, SU5416 directly inhibited the enzyme activity of rat cerebellum nNOS with an IC50 value of 22.7 µM. In addition, knock-down of nNOS expression using short hairpin RNA (shRNA) abolished the neuroprotective effects of SU5416 against MPP+-induced neuronal loss. Our results strongly demonstrate that SU5416 might exert its unexpected neuroprotective effects by concurrently reducing nNOS protein expression and directly inhibiting nNOS enzyme activity. In view of the capability of SU5416 to cross the blood-brain barrier and the safety for human use, our findings further indicate that SU5416 might be a novel drug candidate for neurodegenerative disorders, particularly those associated with NO-mediated neurotoxicity.

The anti-cancer agent SU4312 unexpectedly protects against MPP(+) -induced neurotoxicity via selective and direct inhibition of neuronal NOS.

SU4312, a potent and selective inhibitor of VEGF receptor‐2 (VEGFR‐2), has been designed to treat cancer. Recent studies have suggested that SU4312 can also be useful in treating neurodegenerative disorders. In this study, we assessed neuroprotection by SU4312 against 1‐methyl‐4‐phenylpyridinium ion (MPP+)‐induced neurotoxicity and further explored the underlying mechanisms.